The long range goals of the experiments outlined in this proposal are to understand at the molecular and biochemical level what determines temporal patterns of gene expression during early embryogenesis. It has been made clear from numerous studies that cells committed to different lineages differ in the pattern of genes they express. A fundamental question concerning developmental biologists is to dissect just how these different patterns are generated in the daughters of a single cell, the fertilized egg. Our approach to this problem has been to dissect the cis-acting regulatory sequences and the trans-acting regulatory proteins of families of genes encoding histone H1 proteins that are differentially regulated during early embryogenesis and in adult tissues of the sea urchin. The expression of the early or embryonic histone genes, which are encoded by 300-500 tandem arrays, is confined to a period up to the blastula stage of development about 12 hrs. following fertilization. The late histone gene family consists of 2 single copy genes whose transcripts are expressed from a basal promoter until the blastula stage when their transcription rate increases and about 1 million additional late H1 mRNAs per embryo accumulate during the next 8 hrs. Our experimental approach to the questions outlined above has been to identify the DNA sequences required for the accurate stage-specific initiation of transcription and to purify and characterize the proteins that bind to these sequences. We will test the biological activity of these factors, produce antisera, isolate cDNAs and determine when and where these proteins are present in oocytes, eggs, embryos and adult tissues. This reverse genetic approach has led to the identification of both promoter specific elements and an enhancer element that act as molecular timing switches for stage specific embryonic transcription. For example, the late H1 gene is activated at the mid- blastula stage by an enhancer element that consists of 3 binding sites for a single protein, Stage Specific Activator Protein (SSAP). We have purified SSAP and obtained cDNA clones encoding this protein. SSAP is a novel transcription factor because it can bind to single stranded DNA and it has a transcription activation domain that is 6-10 fold more potent than VP16 in mammalian cells. We hope to understand how this potent transcription activation domain functions as a molecular timing switch by mutagenesis, identification of interacting proteins, and post- translational modification. Since this is such a potent transcription activator, this suggests that it could interact with unique components of the transcription machinery that we hope to identify and isolate. SSAP is related to two human proteins, EWS and TLS, of unknown function except they are involved in chromosomal translocations in Ewings Sarcoma and Liposarcoma respectively. We have identified and have candidate clones for a human homologue of SSAP and we will ask if this homologue is a candidate for involvement in human disease. These studies pertain directly to understanding the precise and detailed mechanisms underlying differential gene expression during embryogenesis and differentiation.